1. Field of the Invention
This invention relates to the design of a double antigenically marked classical swine fever virus (CSFV) live attenuated candidate strain vaccine. The Flag/T4 virus is a recombinant CSFV which contains a positive foreign antigenic marker due to the insertion of the highly antigenic epitope, Flag, into the 19mer insertion of the E1 structural protein of recombinant virus RB-C22, as well as a negative antigenic marker, i.e., changes in the WH303 epitope of the E2 protein which result in the lack of reactivity with monoclonal antibody WH303.
2. Description of the Relevant Art
Classical swine fever (CSF) is a highly contagious disease of swine that can be either acute or chronic in nature (van Oirschot, J. T. 1986. In: Diseases of Swine, 6th edition, Leman et al., eds., Iowa State University Press, Ames, Iowa, page 289). The etiological agent, CSF virus (CSFV), is a small, enveloped virus with a positive, single-stranded RNA genome. Along with bovine viral diarrhea virus (BVDV) and border disease virus (BDV), CSFV is classified as a member of the genus Pestivirus within the family Flaviridae (Becher et al. 2003. Virology 311: 96-104). The 12.5 kb CSFV genome contains a single open reading frame that encodes a 3898 amino acid polyprotein and ultimately yields 11 to 12 final cleavage products (NH2-Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B—COOH) through co- and posttranslational processing of the polyprotein by cellular and viral proteases (Rice, C. M. 1996. In: Fundamental Virology, 3rd edition, Fields and Howley, eds., Lippincott Raven, Philadelphia, pp. 931-959).
CSFV is enzootic in all continents, and actively circulating in South and Central America, southern Mexico, and the Caribbean. Disease outbreaks occur intermittently in Europe where control programs of the disease (Westergaard et al. 1998. In: Vaccines in Agriculture: Immunological Applications To Animal Health and Production, Wood et al., eds., CSIRO, East Melbourne, Australia, pages 13-20) include quarantine and eradication of infected herds, resulting in the elimination of a large number of animals, including noninfected animals, thus engendering significant economic losses. Vaccination, quarantine and testing protocols offer an alternative to these measures and may become the only means to control and eradicate an outbreak of CSF, reducing the economic impact that results from the heretofore elimination of such a vast number of pigs.
Safe and effective CSFV vaccines that prevent clinical symptoms of the disease and virus spreading, including during the early post-vaccination period, have been used around the world (Aynaud, J. M. 1988. In: Classical Swine Fever and Related Viral Infections, B. Liess, ed., Nijhoff, Boston, Mass., pages 165-176; Biront and Leunen. 1988. ibid., pages 181-197). Among available CSFV vaccines, live attenuated vaccines, such as C strain, GPE-strain, Thiversal strain, and PAV-250, confer effective and long lasting immunity against CSF (Biront and Leunen, ibid.). In general, these vaccines have been obtained after serial passages of CSFV isolates in tissue culture or rabbits; however, the genetic bases of the attenuation in the above cases are unknown. Additionally, it is not currently possible to distinguish, serologically, between animals vaccinated with live attenuated vaccines and animals infected with wild-type virus.
Development of infectious CSFV cDNA clones has enabled genetic approaches for defining mechanisms of viral replication and pathogenesis. Infectious clones of the attenuated CSFV C-strain and the pathogenic Alfort/Tübingen strain have been constructed, enabling identification of Ems and Npro as virulence factors in swine and the role of different Ems mutations in virus attenuation (Meyers et al. 1996, supra, Moormann et al. 1996. J. Virol. 70: 763-770, Ruggli et al. 1996. J. Virol. 70:3478-3487, Mayer et al. 2004. Vaccine 22: 317-328). CSFV infectious clones have been used to identify viral proteins or protein domains functioning in viral replication and virulence, and to engineer attenuated marker CSF live attenuated vaccines (Meyers et al. 1999, supra, Moser et al. 2001. Virus Genes 23: 63-68, Tratschin et al. 1998. J. Virol. 72: 7681-7684, van Gennip et al. supra).
The capsid protein, and glycoproteins Erns, E1, and E2 are the structural components of the CSFV virion with E1 and E2 anchored to the envelope by their carboxyl termini and Erns loosely associated with the viral envelope (Thiel et al. 1991. J. Virol. 65: 4705-4712; Weiland et al. 1990. J. Virol. 64: 3563-3569; Weiland et al. 1999. J. Gen. Virol. 80: 1157-1165).
E1 and E2 are type I transmembrane proteins with an N-terminal ectodomain and a C-terminal hydrophobic anchor (Thiel et al., supra). E2, the major structural protein, is considered essential for CSFV replication, as virus mutants containing partial or complete deletions of the E2 gene have proven non-viable (van Gennip et al. 2000. Vaccine 19:447-459). E2 is the most immunogenic of the CSFV glycoproteins (Konig et al. 1995. J. Virol. 69: 6479-6486; van Gennip et al. and Weiland et al. 1990, supra), inducing neutralizing antibodies and protection against lethal CSFV challenge (Wensvoort et al. 1989. Vet. Microbiol. 21: 9-20; Van Zijl et al. 1991. Vaccine 17: 433-440; Hulst et al. 1993. J. Virol. 67: 5435-5442; Rumenapf et al. 1991. J. Virol. 65: 589-597; Van Rijn et al. 1996. J. Gen. Virol. 77: 2737-2745; Van Rijn et al. 1998. Vaccine 17: 433-440). CSFV E2 also contains, between residues 829 and 837, an epitope recognized by monoclonal antibody (mAb) WH303 (Lin et al. 2000. J. Virol. 74:11619-11625), a reagent which is routinely used for CSF diagnostics. WH303 mAb does not react with E2 of BVDV or BDV.
Candidate CSFV subunit marker vaccines have been developed using recombinant E2 envelope protein (Van Zijl et al., supra; Hooft van Iddkinge et al. 1996. Vaccine 14: 6-12; Hulst et al, 1993, supra; Van Rijn et al., supra). Different E2 protein domains have been described as targets for neutralizing monoclonal antibodies (Wensvoort et al., supra), but E2 subunit vaccines have not been found to be as efficacious as traditional live attenuated vaccines, particularly when animals are challenged shortly after vaccination (Hulst et al., 1993, supra; Van Rijn et al., supra; Risatti et al. 2003. J. Clin. Microbiol. 41: 500-505). The failure to induce rapid and efficient protective immunity precludes the use of subunit vaccines as an emergency control measure during a CSFV outbreak. DNA vaccines encoding E2, when expressed, also induced protection in pigs; however, again, rapid elicitation of protection was not proven (Andrew et a. 2000. Vaccine 17: 1932-1938; Yu et al. 2001. Vaccine 19: 1520-1525).
Recently, infectious clone technology has enabled antigenic modification of attenuated CSFV strains for use as experimental marker live attenuated CSF vaccines. Infectious clones of the attenuated C-strain were used to replace the antigenic region of E2 and/or the complete Ems gene with analogous sequences from Bovine Viral Diarrhea Virus (BVDV). Preliminary data indicated that both chimeric viruses were able to induce protection in pigs at one week after vaccination. Significantly, chimera-induced anti-CSFV antibody responses could be discriminated from those produced by parental virus (van Gennip et al., supra).
The development of disease control strategies in the event of a CSFV outbreak requires rapid onset of protection, which becomes a more important parameter of vaccine performance than, for example, duration of protection. The development of such vaccines would imply the production of rationally designed live attenuated vaccine CSFV strains.